Abstract

Background

In Schizosaccharomyces pombe the SET domain protein, Set3p - together with its interacting partners, Snt1p, and Hif2p - form a complex that aids in preventing cell division failure upon mild cytokinetic stress. Intriguingly, the human orthologs of these proteins (MLL5, NCOR2, and TBL1X) are also important for the faithful completion of cytokinesis in tissue culture cells. Since MLL5, NCOR2, and TBL1X form a complex with the histone deacetylase, HDAC3, we sought to determine if an orthologous counterpart played a regulatory role in fission yeast cytokinesis.

Results

In this report we identify the hos2 gene as the fission yeast HDAC3 ortholog. We show that Hos2p physically interacts with Set3p, Snt1p, and Hif2p, and that hos2∆ mutants are indeed compromised in their ability to reliably complete cell division in the presence of mild cytokinetic stresses. Furthermore, we demonstrate that over-expression of hos2 causes severe morphological and cytokinetic defects. Lastly, through recombinase mediated cassette exchange, we show that expression of human HDAC3 complements the cytokinetic defects exhibited by hos2∆ cells.

Conclusions

These data support a model in which Hos2p functions as an essential component of the Set3p-Snt1p-Hif2p complex with respect to the regulation of cytokinesis. The ability of human HDAC3 to complement the cytokinesis defects associated with the deletion of the hos2 gene suggests that further analysis of this system could provide insight into the role of HDAC3 in both the regulation of cell division, as well as other biological processes influenced by HDAC3 deacetylation.

Keywords

Fission yeastCytokinesisCell divisionHistone deacetylase

Background

In the fission yeast, Schizosaccharomyces pombe, regulatory mechanisms exist to ensure that cytokinesis takes place at the correct spatial location within the cell and at the proper temporal position of the cell cycle [1–5]. The proper spatial positioning of the cytokinetic actomyosin ring is controlled by the anilin-related protein, Mid1p, which - upon entry into mitosis - re-localizes from the nucleus to a medial band defining the future site of cell division [5–9]. The initiation of ring constriction, on the other hand, is signalled by a conserved regulatory module referred to as the Septation Initiation Network (SIN). The SIN is composed of a GTPase signalling cascade that is essential for the temporal co-ordination of cytokinesis, ring constriction, and for the deposition of the division septum [3, 5, 9, 10].

In addition to these mechanisms, recent work has also supported the existence of a cytokinesis monitoring system. This system has the capacity to generate a prolonged cytokinesis competent state (characterized by delayed entry into mitosis and the continuous repair/re-establishment of the actomyosin ring) that allows for the successful completion of cell division upon mild cytokinetic stresses [11–17]. The key components of this regulatory module are the Cdc14 family phosphatase, Clp1p, and the 14-3-3 protein, Rad24p. When challenged by stresses that perturb the cell division machinery, a phosphorylated form of Clp1p (normally nucleolar) becomes bound by Rad24p leading to its prolonged retention in the cytoplasm and the extended activation of the SIN. In the absence of either Clp1p or Rad24p, cells are unable to maintain SIN signalling leading to cytokinesis failure and the generation of inviable, multinucleate cells [13, 15, 18].

A useful strategy in both identifying these regulators, and in defining their roles, has involved the treatment of fission yeast cells with the actin depolymerising drug, Latrunculin A (LatA) [11–15]. At the concentrations used (20–50 times less than that needed to completely depolymerize the actin cytoskeleton) such treatment impedes constriction of the actomyosin ring and is lethal to both clp1∆ and rad24∆ mutants (due to their inability to prolong the cytokinesis competent state). Wild-type cells in contrast, are able to complete cell division under these conditions, albeit at rates slower than in untreated cells.

Interestingly, a recent genome-wide genetic screen based on the isolation of deletion mutants hyper-sensitive to LatA, identified set3hif2, and snt1 and showed that their respective gene-products form a nuclear-localized complex required for the dependable execution of cytokinesis. Further analysis demonstrated that set3∆ mutants were unable to properly modulate the expression of stress response genes, suggesting a role for the Set3p complex in effecting changes in gene expression required to counter the effects of LatA induced stress [19].

Intriguingly, the set3snt1, and hif2 genes are orthologous to human MLL5NCOR2 and TBL1X, which together encode components of a histone deacetylase complex. Remarkably, knockdown of either of these genes in human HeLa cells results in increased rates of cytokinesis failure [20]. Since NCOR2, and TBL1X physically associate with the type I histone de-acetylase, HDAC3 - a highly conserved histone deacetylase with orthologs from Dictyostelium to multicellular mammals - we sought to determine if an orthologous counterpart played a regulatory role in fission yeast cytokinesis [20–22].

Here we identify the hos2 gene as the fission yeast HDAC3 ortholog. Hos2p, also known as Hda1p, is a non-essential histone de-acetylase known to affect H4K16 acetylation (primarily in the 5′ end of genes) as well as gene silencing and sporulation efficiency [23–25]. In this report we show that Hos2p exists in a complex with Set3p, Snt1p, and Hif2p, and that hos2∆ mutants are also compromised in their ability to complete cytokinesis in the presence of low doses of LatA. Furthermore, a role in the regulation of cell division is supported by the severe morphological and cytokinetic defects observed upon hos2 over-expression.

Lastly, we provide strong support for the conservation of HDAC3 function by demonstrating the ability of human HDAC3 to complement the cytokinetic defects exhibited by hos2∆ cells.

Results

Hos2p is required for the successful completion of cytokinesis in response to perturbation of the cell division machinery

[20–22] To determine if an ortholog of HDAC3 existed in S. pombe, and if it too played a role in the regulation of cytokinesis, A BLAST search using human HDAC3 as query was performed. This analysis revealed strong conservation of amino acid sequence between HDAC3 and fission yeast Hos2p (not be confused with the DASH complex subunit, Dad2p, which is also sometimes referred to using the gene name, hos2). The proteins share 51% identity (63% similarity), are of similar length (427 and 437 aa, respectively), and possess a single histone deacetylase domain (PFAM00850) that comprises almost the entire length of the protein (Additional File 1).

To determine if Hos2p played a role in cytokinesis, the hos2 gene deletion mutant was purchased from the commercial supplier, Bioneer. After confirmation of the deletion via colony PCR, wild-type and hos2∆ strains were grown to mid-log phase and serial dilutions plated onto YES media containing either 0.5 μM LatA or DMSO (solvent control).

Interestingly, the hos2∆ strain demonstrated a substantial decrease in viability when grown in the presence of LatA. In contrast, while the rate of growth of wild-type cells decreased in LatA media, viability was not affected (note the formation of small colonies even at the lowest dilution) (Figure 1A).

Figure 1

hos2∆andhos2-Y321Hmutants are hyper-sensitive to LatA treatment. (A) Ten-fold serial dilutions of logarithmically growing cells of the indicated genotype were plated onto YES plates containing 0.5 μM LatA or DMSO (solvent control) at 30°C for 3 d. (B) Cells of the indicated genotype were grown to mid-log phase at 30°C and then treated with 0.5 μM LatA for 5 h before being fixed and stained with DAPI (nuclei) and aniline blue (cell wall/septa). Bar, 10 μm. (C) Quantitation of phenotypes of cells treated as in B. Between 200 and 500 cells were counted for each genotypic class.

To determine if the sensitivity to LatA was related to defects in cytokinesis, both wild-type and hos2∆ strains were grown in liquid YES media and then treated with either 0.5 μM LatA or DMSO for 5 hours at 30°C. Cells were then fixed and stained with DAPI and analine blue to visualize nuclei and cell wall/septal material, respectively. No obvious morphological or cytokinesis phenotypes were observed in hos2∆ cells under normal growth conditions. However, in LatA media, hos2∆ mutants were severely impaired in their ability to complete cell division and accumulated a large proportion of tetra-nucleate cells with fragmented septa. In contrast, the majority of wild-type cells were bi-nucleate and formed functional, albeit thickened and sometimes malformed septa (Figure 1B).

To quantitate the data, cells were classified into four different phenotypic categories:i) uni-nucleate cells, ii) bi-nucleate cells with a functional septum (i.e. the septum completely bisects the cell), iii) bi-nucleate cells with a fragmented septum (i.e. the septum is non- functional and does not completely bisect the cell), and iv) tetra-nucleate cells. This analysis revealed that while over 40% of hos2∆ cells were tetra-nucleate, only 6% of wild-type cells showed a similar phenotype. Moreover, while 72% of wild-type cells were either mono- nucleate, or bi-nucleate (with a functional septum), only 31% of hos2∆ cells were similarly distributed (Figure 1C). The phenotypes observed in hos2∆ mutants upon LatA treatment are unlikely to be due to defects in SIN activity since two independent markers of active SIN signalling - Cdc7p localization to a single SPB, and increased export of Clp1p from the nucleolus to the cytoplasm [15] - were normal in hos2∆ mutants upon exposure to LatA (Additional File 2).

An important role for Hos2p in responding to LatA treatment was also supported by synthetic genetic interactions between the hos2∆ and clp1∆ mutations. Since Clp1p is required for the function of the cytokinesis checkpoint, weak cytokinesis mutants often display stronger phenotypes in clp1∆ backgrounds [15]. To test if this were true in the case of hos2∆ mutants, clp1∆hos2∆ and clp1∆ hos2∆ mutants were examined after treatment with both 0.1 μM and 0.5 μM LatA. Interestingly, while hos2∆ and clp1∆ single mutants were viable at 0.1 μM LatA, double mutants displayed severe cytokinesis defects at this concentration (Additional File 3).

Lastly, we also noted that the presence of the hos2∆ mutation was capable of lowering the restrictive temperature of the ts cdc15-140 mutation by ~ 2°C (cdc15 encodes an F-BAR protein required for contractile ring assembly; [26]) (Additional File 4). Interestingly, this decrease in the restrictive temperature of the cdc15-140 mutation is similar to that caused by the presence of the set3∆snt1∆, and hif2∆ gene deletions in cdc15-140 backgrounds. These data further support a common function for the hos2set3snt1, and hif2 genes [19].

Once having established that the Hos2p protein was indeed involved in the regulation of cytokinesis, we explored the possibility that the protein’s deacetylase activity was related to its function. To this end we created a mutant strain expressing a form of Hos2p in which the catalytically active tyrosine residue in the catalytic pocket was replaced with a catalytically inactive histidine residue (Y321H). Interestingly, the cytokinesis phenotypes of this mutant were more severe than those displayed by hos2∆ cells. Cells bearing the Y321H mutation were almost completely inviable in the presence of LatA and furthermore, over 60% were tetra-nucleate after 5 hours growth in liquid media containing 0.5 μM LatA (Figure 1A-C).

To more closely examine the effects of LatA on cytokinesis we created a hos2∆ strain expressing a marker of the actomyosin ring, Rlc1-GFP [27]. Using the CellAsics ONIX Microfluidic Perfusion Platform, we were able to monitor the constriction of the actomyosin ring while perfusing liquid YES media containing either 0.5 μM LatA or DMSO as a solvent control. In DMSO media, wild-type cells were able to fully constrict the ring in approximately ~25 minutes. Similarly, rings in hos2∆ mutants grown in DMSO media displayed comparable kinetics and were also able to fully constrict in ~25 minutes (Figure 2, top two rows; Additional Files 5 and 6). However, when grown in LatA media, hos2∆ cells displayed dramatic differences in phenotype compared to wild type. The majority of wild-type cells were able to form and constrict the ring in the presence of LatA, albeit over a much longer time frame (~90 minutes) than DMSO controls (7 out of 9 cells). The remainder (2 out of 9 cells) were able to maintain the integrity of the ring over this time frame, but were not able to fully constrict the ring over the 90 minute time-lapse. In contrast, hos2∆ cells could not preserve the physical integrity of the ring. In LatA treated hos2∆ mutants, Rlc1- GFP signal did not constrict and instead appeared to fragment (8 out of 8 cells) within 10–15 minutes (Figure 2, bottom two rows; Additional Files 7 and 8).

Figure 2

hos2∆cells are unable to maintain the integrity of the actomyosin ring upon LatA treatment. Wild-type and hos2∆ cells expressing the Rlc1-GFP fusion protein (marker of the actomyosin ring) were imaged upon treatment with 0.5 μM LatA or DMSO using the CellAsics ONIX Microfluidic Perfusion Platform. Images were taken every 30 s. Arrow indicates fragmented ring.

To explore whether Hos2p played a dosage dependent role in cytokinesis we decided to test the effects of hos2 over-expression using the pREP series of thiamine repressible expression vectors [28, 29]. To this end, full length hos2 was cloned downstream of the nmt1/41/81 promoters present within the pREP1/41/81 plasmids and the constructs were transformed into wild-type strain, JK484 (Table 1). Full strength expression is obtained from the nmt1 promoter, while nmt41 and nmt81 promoters contain site mutations that decrease expression to intermediate and low levels, respectively [28].

Table 1

Strains used in this study

Strain Name

Relevant Genotype

Source

JK9

clp1::ura4+ura4-D18 h-

JK Collection

JK468

hos2::KanMX4 ura4-D18 leu-32 ade6-216

Bioneer

JK484

ura4-D18 leu‐32 ade6‐216 his3-D1

JK Collection

JK561

hos2-GFP::ura4+ura4-D18 leu1-32 ade6- 210 his3-D1

This Study

JK648

hos2-Y321H::ura4+ura4-D18

This Study

JK659

rlc1GFP::ura4+hos2::kanMX4 ura4-D18

This Study

JK737

hos2::ura4+ ura4-D18 leu‐32 ade6‐216 his3-D1 (base strain)

This Study

JK744

hos2::hos2Spura4-D18 leu‐32 ade6‐216 his3-D1

This Study

JK745

hos2::HDAC3Hsura4-D18 leu‐32 ade6‐21 6his3-D1

This Study

JK759

hos2::kanMX4 clp1::ura4+ura4-D18

This Study

JK761

hos2::kanMX4 cdc15-140 ade6-21x leu1-32

JK Collection

JK776

cdc7GFP::ura4+hos2::ura4+ura4-D18

This Study

JK778

clp1GFP::kanMX4 hos2::ura4+ ura4-D18

This Study

MBY154

cdc15-140 ade6-21x leu1-32 h-

JK Collection

MBY624

rlc1GFP::ura4+ura4-D18 h+

JK Collection

MBY978

clp1GFP::kanMX4 ura4-D18 leu1-32 ade6-216 h+

JK Collection

MBY2415

cdc7GFP::ura4+ura4-D18 ade6-21x leu1-32 ura4-D18 his3-D1 h+

JK Collection

SCG5

hos2-myc::ura4+ura4-D18 leu1-32 ade6-210 his-D1

This Study

SCG10

set3-HA::ura4+hos2-myc::ura4+ura4-D18

This Study

SCG11

snt1-HA::ura4+hos2-myc::ura4+ura4-D18

This Study

SCG14

hif2-HA::ura4+hos2-myc::ura4+ura4-D18

This Study

In the presence of thiamine (repressed) cells expressing hos2 from either nmt41, or nmt81 promoters (as well as an empty vector control) displayed normal growth whereas cells expressing hos2 from the nmt1 promoter showed slight growth inhibition (most likely due to the fact that expression from nmt1 is somewhat “leaky”) [28, 29]. On the other hand, when grown in the absence of thiamine (de-repressed) cells expressing hos2 displayed an inhibition of growth ranging from severe (in the case of the nmt1 promoter) to intermediate (in the case of the nmt41 promoter) to mild (in the case of the nmt81 promoter) (Figure 3A, B).

Interestingly, expression of hos2 from the nmt1 promoter also led to a series of unusual and pleiotropic phenotypes related to morphogenesis and/or cytokinesis. While the majority of cells appeared normal, others displayed phenotypes ranging from i) slight morphological abnormalities such as a rounded, de-polarized appearance, ii) misplaced, but otherwise normal septa, iii) abnormally excessive and mis-localized septal deposition, iv) multiple septa v) highly elongated cells containing multiple aberrant deposits of septal material and vi) highly unusual branched and elongated cells (Figure 3C; Table 2). This broad range of phenotypes further supports a model where Hos2p plays a role in the regulation of cytokinesis and/or morphogenesis.

Table 2

Quantitation of phenotypic abnormalities uponhos2orHDAC3over-expression from thenmt1promoter in the absence of thiamine

Morphological Abnormality

pREP1

pREP1-hos2

pREP1-HDAC3

Normal

> 99%

52%

54%

Rounded/Depolarized

< 1%

26.5%

29%

Misplaced septa

<1%

9%

5%

Excessive/Misplaced septal deposits

0

6%

6%

Multiple septa

0

2%

2%

Elongated with multiple aberrant deposits of septal material

0

3%

2%

Elongated and Branched

0

1.5%

1.5%

Hos2p is nucleo-cytoplasmic and physically interacts with Set3p, Snt1p, and Hif2p

We have previously shown that the Set3p, Snt1p, and Hif2p form a nuclear-localized complex. If indeed a member of this complex, Hos2p would be expected to localize (at least in part) to the nucleus. To test this prediction we created a strain expressing a C- terminal Hos2-GFP fusion protein under control of the native hos2 promoter. Hos2p localized to both the cytoplasm and to the nucleus as judged by co-staining with the nuclear dye, DAPI (Figure 4A). Furthermore, we determined that the intracellular distribution of Hos2p was not affected by LatA treatment (data not shown), nor did its localization change as a function of cell cycle position (Figure 4A). The localization of the Scw1p protein (enriched in the cytoplasm relative to the nucleus) was used as a control to ensure the validity of the observed nuclear signal (Figure 4A, bottom panels) [30].

Figure 4

Hos2p localizes to both the cytoplasm and the nucleus and physically interacts with Set3p, Snt1p, and Hif2p.(A) Cells expressing the Hos2-GFP fusion protein were grown to mid-log phase at 30°C in YES media, fixed, and then stained with DAPI and observed using the DAPI and GFP filter sets. Bar, 5 μm. (B) Cells expressing the indicated fusion proteins were grown to mid-log phase in YES, lysed under native conditions, and subjected to anti-HA immunoprecipitations. Both total lysates and immunoprecipitates were resolved by SDS-PAGE and immunoblotted with antibodies specific for the Myc epitope.

To determine if Hos2p was able to physically interact with members of the Set3p complex, in-vivo co-immunoprecipitation experiments using Myc or HA epitope tagged alleles were performed. Significantly, Hos2-Myc fusion proteins could be detected in anti- HA immuno-precipitates of Snt1-HA Hos2-Myc, Hif2-HA Hos2-Myc, and Set3-HA Hos2- Myc extracts, but not in extracts expressing only Hos2-Myc (Figure 4B). These results are consistent with high throughput proteomics experiments aimed at defining fission yeast protein complexes related to histone modification [31]. Taken together, these data suggest that Hos2p molecules can indeed exists within a complex with Snt1p, Hif2p, and/or Set3p.

To further explore the possibility of functional conservation between human HDAC3 and fission yeast Hos2p, we utilized the technique of recombinase mediated cassette exchange to replace the endogenous Hos2p open reading frame in fission yeast with the human HDAC3 gene (Additional File 9) [32]. This approach first required the creation of a “base strain” in which the hos2 gene was replaced with a deletion cassette composed of the ura4+ selectable marker flanked by the Cre recombinase recognitions sites, loxP and loxM3. As expected, the hos2∆ “base strain” created in this manner was indistinguishable from the Bioneer gene deletion mutant in terms of its sensitivity to LatA and its ability to complete cytokinesis in liquid LatA media (Figure 5A-C).

Figure 5

Expression of humanHDAC3complements the cytokinesis defects exhibited byhos2∆mutants. (A) Ten-fold serial dilutions of logarithmically growing cells of the indicated genotype were plated onto YES plates containing 0.5 μM LatA or DMSO (solvent control) at 30°C for 3 d. (B) Cells of the indicated genotype were grown to mid-log phase at 30°C and then treated with 0.5 μM LatA for 5 h before being fixed and stained with DAPI (nuclei) and aniline blue (cell wall/septa). Bar, 10 μm. (C) Quantitation of phenotypes of cells treated as in B. Between 200 and 500 cells were counted for each genotypic class.

Next, we cloned the HDAC3 cDNA from the pOTB7 plasmid (purchased from Open Biosystems) into the XhoI and SacI sites of the “exchange” plasmid pAW8X. The exchange plasmid contains the Cre recombinase gene under control of the nmt41 promoter. Once transformed into the base strain, expression of the recombinase permits exchange of the ura4+ cassette with the HDAC3 sequence (Additional File 9). In this way the hos2::HDAC3Hs (Hs, Homo sapiens) strain - in which the human HDAC3 gene is under control of the S. pombe hos2 promoter - was created. In addition a control strain, in which the S. pombe hos2 gene was re-engineered back into the base strain, hos2::hos2Sp (Sp, Schizosaccharomyces pombe) was used as a control.

Remarkably, expression of human HDAC3 in S. pombe was able to both restore viability and substantially complement the cytokinesis phenotypes characteristic of the base strain, albeit not to the same extent as the re-introduced hos2 gene (Figure 5A-C). While the base strain displayed over 50% tetra-nucleate cells after 5 hours, the hos2::hos2Sp strain displayed only 5% tetra-nucleate cells and the hos2::HDAC3Hs strain only 18% tetra-nucleate cells. Thus, expression of the human HDAC3 is indeed capable of at least partially complementing the loss of the S. pombe hos2 gene.

Lastly, to provide further evidence of conserved function, we proceeded to over- express human HDAC3 under the control of the nmt1 promoter using the pREP1 plasmid. Similar to the over-expression of hos2, over-expression of HDAC3 resulted in a decrease in growth rate in the absence of thiamine (de-repressed) and similar morphological and cytokinesis phenotypes (compare Figure 3B,C with Figure 6B,C). Just as with the case of the hos2 gene, over-expression of HDAC3 resulted in a similar range of pleiotropic phenotypes including i) cells with a rounded, de-polarized appearance, ii) misplaced septa, iii) abnormally excessive and mis-localized septal deposition, iv) multiple septa v) highly elongated cells containing multiple aberrant deposits of septal material and vi) elongated and branched cells (Figure 6B,C; Table 2). Taking all data together, these results suggest that Hos2p and human HDAC3 function in a similar manner and modulate similar biological processes in S. pombe.

Discussion

While the absence of cytokinesis is tolerated under certain specialized circumstances (e.g. the development of Drosophila embryos) it is normally essential for cellular proliferation, differentiation and for maintaining control over ploidy [1, 2, 33]. Thus, knowledge of the regulatory networks governing cytokinesis is an important component of our basic understanding of eukaryotic cell biology. Moreover, aspects of this knowledge related to cytokinesis failure may also be relevant to our understanding of the mechanisms important for maintaining genomic integrity.

Interestingly, a recent genome-wide RNAi screen, aimed at discovering gene-products important for the fidelity of cell division in HeLa cells, identified physical interactors of HDAC3 as being important for cytokinesis [20]. In this work Kittler et al., showed that knockdown of MLL5NCOR2, or TBL1X (genes encoding putative chromatin-binding proteins implicated in transcriptional regulation) resulted in increased rates of cytokinesis failure and the generation of tetraploid intermediates [20]. Remarkably, these genes are orthologous to three fission yeast genes (set3snt1, and hif2) recently identified as playing a role in the reliable execution of cytokinesis and which physically interact with the S. pombe HDAC3 ortholog, Hosp2p (Figure 4B). These results not only further validate the use of S. pombe as a eukaryotic model, but also raise the intriguing question of whether these orthologous complexes represent an evolutionarily conserved module important for the faithful execution of cell division.

In this report we identify the fission yeast ortholog of HDAC3 and provide further evidence of cross-species conservation. First, the expression of human HDAC3 clearly complements the cytokinesis defects displayed by hos2∆ mutants (Figure 5). Second, over- expression of Hos2p or HDAC3 both result in phenotypes related to morphogenesis and/or cytokinesis (Figure 6). With respect to functional conservation, it is also important to note the similarity in localization between Hos2p and HDAC3. HDAC3 is the only member of the class I HDACs to localize to the cytoplasm, as well as the nucleus, owing to the presence of both nuclear import and export signals [38]. Thus, our observation that Hos2p localizes to both of these intracellular compartments represents another level at which Hos2p and HDAC3 share similarities (Figure 4A). Furthermore, the cytoplasmic localization observed for Hos2p is consistent with immunofluorescence data demonstrating that HA epitope tagged Hos2p is predominantly cytoplasmic as well as with global GFP-fusion based localisation studies showing both nuclear and cytoplasmic localization [39, 40].

We also provide genetic evidence supporting a catalytic role for Hos2p through the analysis of strains bearing the Y321H mutation. The mutated tyrosine residue catalyzes stabilization of the transition state between acetyl-lysine and lysine thereby allowing for catalysis of lysine deacetylation; this is not chemically possible through a histidine residue [41]. As expected if Hos2p played a catalytic role, hos2-Y321H mutants displayed phenotypes similar to those exhibited by hos2∆ strains. In fact, the severity of the cytokinesis defects was greater in the site-mutant compared to the gene deletion (Figure 1). We speculate that the increase in severity of the phenotype may be related to the presence of the mutant protein interfering with other components of the pathway in a dominant negative fashion.

While targeted deacetylation is likely an important aspect of Hos2p function, the physiological substrates of the Hos2p-Set3p-Snt1p-Hif2p complex remain unknown. One possibility is that cytokinetic failure is related to transcriptional defects stemming from the abnormal acetylation of histones. A role in transcription is supported by the observation that set3∆ mutants are compromised in their ability to alter the expression of stress response genes upon LatA treatment. Thus, cell division failure may be a manifestation of the inability of the mutants to properly counter the effects of LatA induced stress leading to the direct and/or indirect effects on the function of the cytokinetic machinery. When considering such models it should also be noted that the substrate specificity of HDACs is not restricted to histones. In fact, many non-histone substrates, including transcription factors, signalling molecules, and nuclear import factors have been identified as targets [42]. Thus, the possibility exists that the complex may function through both the modulation of transcription and/or the post modification of non-histone targets.

Finally, while highly speculative, it is interesting to note that the liver specific deletion of HDAC3 results in hepatocellular carcinoma in mice [43]. In this work, 20 out of 20 HDAC3−/− displayed low grade hepatocellular carcinoma at a mean age of 10.2 months. Although a role for cytokinesis failure in this phenotype has not been examined, it is interesting to speculate as to whether it played any role in tumour development in this context. Regardless, the demonstration of functional conservation between HDAC3 and Hos2p suggests that further analysis of these proteins in fission yeast might translate into a theoretical framework for understanding the role of HDAC3 in both the regulation of cytokinesis as well as other biological processes influenced by HDAC3 deacetylation.

Conclusions

In this work we demonstrate a role for the histone deacetylase, Hos2p, in promoting the faithful and dependable execution of cytokinesis in fission yeast. Analysis of catalytically inactive hos2-Y321H mutants suggests that its role in this process is mediated through its deacetylase activity. In addition, co-immunoprecipitation experiments indicate that Hos2p regulates cytokinesis as part of a complex with Set3p, Snt1p, and Hif2p (all of which play a documented role in preventing cell division failure). Lastly, the ability of human HDAC3 to complement the cytokinesis defects exhibited by hos2∆ mutants suggests that continued analysis of this system could translate into a theoretical framework for understanding how HDAC3 functions in more developmentally complex organisms.

Methods

Yeast methods

All Schizosaccharomyces pombe strains used in this study are listed in Table 1. Strains were derived from the Karagiannis lab collection, constructed during the course of this work, or purchased from Bioneer Corporation (Alameda, CA). S. pombe cells were cultured in YES or in Edinburgh Minimal Media (EMM) supplemented with adenine, histidine, leucine, and/or uracil. Liquid cultures were grown with shaking (200 rpm) at 30°C [44]. In experiments involving Latrunculin treatment, S. pombe cells were grown to mid log phase (O.D. 0.2) and treated with 0.1-0.5 μM of Latrunculin A (Enzo Life Sciences International, Plymouth Meeting, Pennsylvania) dissolved in DMSO. Cells were grown at 30°C with shaking at 200 rpm for 3–6 hrs, before being fixed with ethanol and stored in PBS pH 7.4. All experiments were repeated a minimum of three times. Plasmid vectors were transformed into S. pombe using the lithium acetate protocol according to Forsburg and Rhind [44].

Plasmid bearing strains were grown in supplemented EMM + thiamine overnight, washed three times with EMM media, and then cultured for 24 hrs. Cells were then fixed with ethanol, stained with DAPI and analine blue, and imaged using a Leica DMI6000B microscope driven by Metamorph software using the DAPI filter set.

To determine the effects of HDAC3 over-expression, the full length human HDAC3 cDNA, was PCR amplified (forward primer: 5′-GAT CGA TTA ATA TGG CCA AGA CCG TGG CC-3′; reverse primer: 5′-CGA TCG GAT CCT TAA ATC TCC ACA TCG CTT T-3′) from the pOTB7 plasmid (Open Biosystems) and cloned into the NdeI and BamHI sites of the pREP series of plasmids. The plasmids were then transformed into JK484 (Table 1) and the effects of over-expression examined as described above.

Authors’ information

At the time of their contributions CG and SR were MSc candidates at the UWO Department of Biology. CG is currently a PhD candidate at the University of Alberta. SR is currently a PhD candidate at McGill University. JH is a BSc (Hons) student at the UWO Department of Biology. JK is an Assistant Professor at the UWO Department of Biology.

Abbreviations

HDAC:

Histone Deacetylase

SIN:

Septation Initiation Network

YES:

Yeast Extract Supplements

EMM:

Edinburgh Minimal Media

LatA:

Latrunculin A

DMSO:

Dimethyl sulfoxide

DAPI:

4′,6-diamidino-2-phenylindole

PBS:

Phosphate Buffered Saline.

Declarations

Acknowledgements

This work was supported by the National Sciences and Engineering Research Council of Canada, the University of Western Ontario Academic Development Fund, and the Canada Foundation for Innovation. We would also like to thank members of the UWO Biology and Biochemistry Departments for helpful discussions and/or critical reading of the manuscript.

13008_2011_164_MOESM9_ESM.tiffAdditional file 9:
Schematic representation of recombinase-mediated cassette exchange. A “base-strain” is first created in which the hos2 open reading frame is replaced with a deletion cassette composed of the ura4+ selectable marker flanked by the Cre recombinase recognitions sites, loxP and loxM3. Next, the “exchange” plasmid (pAW8X-HDAC3) containing the Cre recombinase gene under control of the nmt41 promoter is transformed into the base strain. Expression of the recombinase permits exchange of the ura4+ cassette with the HDAC3 sequence. (TIFF 332 KB)

Below are the links to the authors’ original submitted files for images.

Copyright

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